Summary

Gastric microbiology of the pig has been given a great deal
of attention in the past decade. Much of the work in this area
has been published in human medical journals, and therefore, swine
practitioners may not be aware of these recent findings. The pig
has been used in the study of gastroduodenal ulcers of humans.
The stomach of pigs has been successfully colonized experimentally
by Helicobacter pylori, the bacterium associated with gastric
ulceration and gastritis in humans. Although H. pylori
does not naturally inhabit the pig's stomach, there are spiral-shaped
bacteria that can commonly be found in the porcine gastric mucosa.
This bacterium has been tentatively named Helicobacter heilmannii
(formerly Gastrospirillum suis). Helicobacter heilmannii
is found in several other species, including dogs, cats, and occasionally
humans. Survey studies show that pig contact is an important risk
factor for humans becoming infected with H. heilmannii.
Infection of humans with H. heilmannii causes a milder
form of chronic gastritis than H. pylori and may be self-limiting.
At the present time, it is unclear whether H. heilmannii
infection of swine causes disease. In swine, the site of ulceration
is in the pars oesophagea whereas H. heilmannii colonizes
the fundic and pyloric regions. Pigs experimentally infected have
not developed gastric ulcers; however, several epidemiological
studies have found a strong association between the presence of
H. heilmannii and ulceration of the pars oesophagea. Practitioners
need to consider this organism as a potential pathogen and a potential
zoonotic bacterium.

Keywords: swine,
Helicobacter, ulcers

Received: February 18, 1999Accepted: April 16, 1999

The field of gastric microbiology has
made rapid advances in the past decade as a result of the discovery
that gastroduodenal ulceration in humans is primarily a result
of a bacterial infection. Research has led to an increase in our
knowledge of the curved or spiral bacteria that inhabit the mucus
layer of the stomach of humans and various animal species, including
swine.

This paper is intended to provide the swine practitioner with
an overview of the numerous publications on Helicobacter
infection, with particular emphasis on its possible role in the
development of gastric ulcers in swine, and its potential zoonotic
spread from pigs to humans.

History

The presence of spiral bacteria in the stomachs of certain
animals has been recognized for over 100 years,1,2
and in the stomachs of humans for at least 60 years.3
Little interest was shown in these micro-organisms until the 1970s,
when a link between the presence of intragastric bacteria and
peptic ulcers in humans was suggested. By the early 1980s, the
association between the presence of bacteria and the occurrence
of chronic gastritis and peptic ulcers was established.4
The species of gastric bacteria that has now become widely accepted
as the major cause of chronic gastritis, gastroduodenal ulceration,
and a major risk factor for gastric cancer in humans has been
identified as Helicobacter pylori.5 As a result
of the discovery of a bacterial etiology, the approach to the
treatment of human gastric ailments has radically changed. The
treatment of gastroduodenal ulcers is accomplished with a short
course of antibiotics in combination with acid-inhibiting drugs.
The success rate of this regimen is very high.6

Researchers have used the pig as a model to facilitate the
study of H. pylori. Gnotobiotic and conventional pigs have
been successfully colonized with experimental infections of H.
pylori.7 In addition, a naturally occurring, spiral-shaped
bacterium (morphologically distinct from H. pylori) has
been identified in the gastric mucosa of commercially-reared pigs.8

It has become apparent that H. pylori is almost exclusively
a human pathogen. However, a second Helicobacter (H.
heilmannii) which infects humans at a much lower prevalence
than H. pylori9 is likely the same species of
Helicobacter found in the stomachs of pigs, as well as
cats and dogs. Therefore, there is a strong possibility that humans
can become infected with H. heilmannii from pigs.10

Bacterial characteristics and nomenclature

Helicobacter resemble Arcobacter and Campylobacter
and share many characteristics. Microbiologists have grouped Helicobacter,
Arcobacter, and Campylobacter within Superfamily
VI, with Arcobacter and Campylobacter forming the
family Campylobacteriaceae. Members of Superfamily VI tend to
be difficult to culture and to define using conventional laboratory
tests. Only recently, using advanced molecular and DNA technology,
has classification been possible.11 Whether or not
a bacterium belongs in the Helicobacter genus has been
based mainly on determining the sequence of bases in its 16S ribosomal
RNA molecule.12 Bacteria with more than 90% homology
in the sequence of bases indicates a close relationship. These
criteria have been used to group bacteria with remarkably different
morphological structure together in the genus Helicobacter.

All Helicobacter are gram-negative and micro-aerophilic.
Commonly, they have multiple polar flagella that are always sheathed
and exhibit urease, catalase, and oxidase activity.12

Two Helicobacter species isolated from human stomachs
differ morphologically from each other. Helicobacter pylori
(originally named Campylobacter pyloridis) are curved or
S-shaped, with flagella at one end. They measure about 4 µm
in length. The second, less prevalent organism, Helicobacter
heilmannii (previously named Gastrospirillum hominis)
is a tightly spiraled bacterium, with the number of turns varying
between four and six. These bacteria, at 7-10 µm in length,
are more than twice as large as H. pylori.13

The spiral bacterium found in the stomach of pigs was originally
called Gastrospirillum suis because of the morphological
similarity to the spiral bacterium "Gastrospirillum hominis"
and other "Gastrospirilla" found in a variety
of animal species. A comparison of the ribosomal RNA between the
human and swine "Gastrospirilla" was shown to
be 99.5% homologous, supporting the hypothesis that the bacterium
of pigs and of humans belongs to the same species.14 Therefore,
recent studies refer to this bacterium in swine stomachs as H.
heilmannii.

So far, more than 13 species of Helicobacter have been
identified in a wide range of omnivorous or carnivorous animals.
Helicobacter heilmannii and H. felis are the two
species most often identified in dogs and cats.15 Not
all Helicobacter species are restricted to the stomach.
Certain Helicobacter species have been identified in the
lower bowel, liver, and in aborted fetuses.15

Epidemiology

Helicobacter pylori infection is widespread, with one-third
to one-half of the world's human population carrying the bacterium.16,17
In developed countries, children are rarely infected but the prevalence
rises with age, so that more than half of all 60-year-olds are
infected. The incidence in developed countries is low (0%-5% per
year), but once acquired, H. pylori infection persists
for years and often the infected individual remains a carrier
for life. In contrast, the prevalence of H. pylori in developing
countries is very high (generally two-thirds of the adult population
infected) and children are commonly infected.6 General
hygiene and sanitation are believed to be important in controlling
the spread of H. pylori. Studies have shown that H.
pylori exists in a higher prevalence in saliva than in feces,18
suggesting that the oral-oral route of spread may be the most
likely means by which H. pylori is transmitted in developed
countries. Fecal transmission is, however, a likely method of
spread when sanitation is poor. An animal reservoir as a source
of H. pylori has not been found, and therefore, person-to-person
contact is considered the primary method of transmission.

Helicobacter heilmannii appears to be widely distributed
among cats, dogs, and pigs. In humans, H. heilmannii is
far less prevalent than H. pylori. About 1% or less of
the human population is believed to be infected with H. heilmannii.19,20
Contact with animals, particularly pigs, has been shown to be
a risk factor to becoming infected with H. heilmannii.10,19
It has been suggested that H. heilmannii infection in humans
may often be self-limiting compared to dogs, cats, and pigs, in
which the bacterium appears to persist.10

The prevalence of H. heilmannii in the commercial pig
population is unknown. In a study of 85 pigs from an Italian slaughterhouse,
9.4% of the stomachs were found to be positive for H. heilmannii
based on histologic examination.21 In an earlier study,
Brazilian researchers reported a similar prevalence, identifying
10.8% of 120 pig stomachs as positive.22 More sensitive
tests for detecting H. heilmannii are being developed and
it is certain that the prevalence rate will be found to be much
higher than in these initial studies.23

Transmission of H. heilmannii is likely via oral-oral
and fecal-oral route from pig to pig. Cross-species spread appears
likely, and therefore, animal vectors may be important in the
introduction of H. heilmannii to a näive swine herd.
In a recent Italian survey of wild rats, histologic evidence of
a spiral bacterium morphologically similar to H. heilmannii
was found in 23% of the animals examined.24 Cats and
dogs are also known carriers of H. heilmannii and need
to be considered as potential vectors.

Pathogenesis

The success of Helicobacter organisms is related both
to unique maintenance factors (allowing the organism to persist
in a hostile environment), and pathogenic mechanisms (leading
to disruption of the gastric mucosal barrier).25 The
most important maintenance (virulence) factors include motility,
adaptive enzymes and proteins, and the ability to adhere to gastric
mucosal cells and mucus. Pathogenic mechanisms include enzymes
and toxins which act as mediators of inflammation or contribute
to acid/pepsin activity.25 The characteristics of Helicobacter
which make them successful colonizers of the stomach are much
better understood than the factors that lead to gastritis and
ulceration.26

Motility has been shown to be an essential component of Helicobacter's
success in colonizing the stomach. Motility is achieved by the
spiral shape and the polar flagella. For an organism to survive
in the stomach, the bacteria must be able to avoid being flushed
away during gastric contractions and stomach emptying. Helicobacter
are able to quickly burrow into the mucus and away from the acid
environment of the stomach lumen. Research using gnotobiotic pigs
has shown that a strain of H. pylori with poor motility
was unable to colonize the pig's stomach but a highly motile strain
was successful.27

Urease production also appears to be an important survival
mechanism. Urease breaks down urea to produce ammonia, which in
turn, helps to neutralize the stomach acid in the surrounding
area. Helicobacter strains exhibiting weak urease activity
failed to colonize in pigs that were experimentally challenged.28
Once colonization has taken place, and the bacterium is located
in the more friendly alkaline environment adjacent to the epithelial
surface, urease production may not be needed as an environmental
modifier but may be an essential component in the pathogenic mechanism.25
Catalase, another important enzyme produced by Helicobacter,
protects the bacterium from the toxic effects of reactive oxygen
metabolites formed in neutrophils from hydrogen peroxide.26

Helicobacter spp. can inhibit acid secretion from isolated
rabbit parietal cells29 and it has been suggested that
specific inhibition of acid secretion may facilitate acute H.
pylori infection. A hypochlorhydria is frequently observed in
human patients recently infected with H. pylori.25

Colonization of H. pylori requires the ability of the
organism to closely adhere to the gastric epithelial surface,
similar to enterotoxigenic E. coli in the small intestine.
Helicobacter heilmannii does not adhere to mucosal cells,
but appears to rely on its very active motility30 to
colonize the mucus and mucosal surface.

An amazing aspect of H. pylori is the fact that a strong
antibody response occurs as a result of infection, and yet the
bacterium generally persists for years, possibly for the life
of the human host. Helicobacter pylori is obviously capable
of evading the human immune mechanisms. This evasion is partly
a result of the inaccessibility and motility of the bacterium,
but other factors are likely to play a role.26 Typically,
the inflammatory reaction occurs deep in the gastric mucosa some
distance from the bacterium, which may explain how the organisms
survive.

A second aspect of bacterial persistence that puzzles researchers
involves nutrient sources. It is unlikely that Helicobacter
spp. could persist for years relying on the food that the host
ingests.6 Therefore, some regulated interaction between
the host cells and bacteria must exist. Possibly Helicobacter
trigger inflammation as a means of acquiring nutrients. Helicobacter
pylori initiates irritation of the epithelia with the release
of urease and production of ammonia. In addition, cytotoxins have
been identified which are directly associated with the degree
of gastritis in gnotobiotic pigs27 and in humans. Helicobacter
pylori strains which produce a toxin inducing the formation
of vacuoles in tissue cultures are 30%-40% over-represented in
ulcer patients compared to those with gastritis alone.6
The gene responsible for toxin production has been identified
and named vacA. A second gene of H. pylori that is associated
with pathogenicity has been sequenced and named cagA. About 50%
of patients with chronic gastritis alone are infected with cagA
strains of H. pylori, but virtually all patients with duodenal
ulcers are infected with cagA strains.6

In humans, Helicobacter infect and inflame the tissue
that becomes ulcerated and therefore one can speculate that direct
insult from the bacteria leads to ulcerative lesions; at least
this appears to occur in certain patients in combination with
predisposing factors. In swine, ulceration is almost exclusively
restricted to the nonglandular pars oesophagea region, whereas
H. heilmannii are observed in the oxyntic and pyloric mucosa.
Therefore, it has been suggested that if Helicobacter is
partially responsible for gastric ulceration in swine, the mechanism
might be that infected stomachs secrete too much acid.13
It has been suggested that hyperacid secretion due to increased
gastrin release is stimulated by the ammonia produced as a result
of H. pylori colonization.31 Supporters of this
theory argue that effects by the bacteria to create a neutral
pH in the layer overlying the gastric epithelium interferes with
the normal inhibition of gastrin release by intraluminal acid.
Eradication of H. pylori generally results in a decrease
in basal and meal-stimulated gastrin secretion.32Helicobacter
heilmannii infections have not been as thoroughly studied
as H. pylori, but at least one study of pigs experimentally
infected showed no difference in gastric pH values before infection
with H. heilmannii compared to after infection.33

In all likelihood, the mechanism responsible for ulceration
of the pars oesophagea in pigs is very different from the pathogenesis
of the human peptic ulcer, considering the major differences in
gastric anatomy and physiology between the two species. Therefore,
any attempt to extrapolate information from studies of H. pylori
in humans to explain gastric ulceration in swine should be viewed
with caution.

Diagnosis

Whereas there are many methods available for diagnosing H.
pylori infection,34,35 detecting H. heilmannii
poses some difficulty.12

In the case of H. pylori diagnosis in humans, tests
can be divided into those requiring endoscopy to gain diagnostic
material and those that are noninvasive and can be performed on
saliva, serum, or breath samples.36 Histologic examination
of biopsy material from the antrum is commonly performed because
the likelihood of finding bacteria in positive cases is high,
especially if more than one sample is taken. In addition, evidence
of inflammation and mucosa pathology can be assessed.34

Often a rapid urease test is performed in conjunction with
histologic examination. This test is not specific to H. pylori
because all Helicobacter species are potent producers of
urease. The test involves placing biopsy material in a urea broth
which contains a pH indicator, such as phenol red. The pH will
rise if urease is present to split urea and create ammonia. If
only a small number of bacteria are present in the biopsy sample,
a color change may not occur, giving a false negative. The test
is only 70%-90% sensitive, but it provides a rapid answer and
is relatively inexpensive to perform.15

Other tests in human medicine that can be performed--using
endoscopically obtained biopsy material--include polymerase chain
reaction (PCR) and culture. Helicobacters tend to be difficult
to grow, making culturing the least sensitive of these tests;
as a result, this is seldom performed outside research institutes.35

Helicobacter pylori gastritis causes a systemic and
local immune response which has been used as the basis for serological
tests. Enzyme-linked immunosorbent assay (ELISA) tests are 95%
sensitive and specific.36,37 Antibodies remain detectable
for 4-6 months after H. pylori eradication.

The urea breath test relies on the ability of Helicobacter
spp. to break down urea that is radiolabeled with C13 or C14 to
produce radioactive carbon dioxide.38 Exhaled air is
sampled and the degree of radioactivity measured as a means of
determining whether urease-producing bacteria are present in the
stomach of the patient. The test is highly sensitive and specific,
easy to perform, and produces a rapid result. The breath test
is commonly used in human medicine as a follow-up method of monitoring
the success of H. pylori eradication programs39
and can be used as a screening procedure to reduce the necessity
of invasive techniques.40

Diagnosing H. heilmannii infection in swine has proven
more difficult. In situ mapping of urease-positive areas is a
very useful technique that can help detect this pathogen.41
This method involves collecting pig stomachs at the abattoir,
opening and gently washing the mucosal surface, and painting a
thin layer of a urea medium containing a pH indicator (phenol
red) over the entire inner stomach surface. Within 2 hours, a
striking color change from yellow to pink indicates areas of urease-producing
bacterial colonies. Distribution of H. heilmannii colonies
has been reported to be patchy, resulting in false negative results
from biopsy examination. The use of a urea mapping technique helps
to overcome this problem. It can also be used as an indirect method
of monitoring for the presence of H. heilmannii on a herd
basis.

Culturing of H. heilmannii has generally been unsuccessful.41,42
Frustrated by an inability to grow non-H. pylori spiral
bacteria in laboratory media, researchers looked for alternative
methods of culture. Mice have been inoculated with gastric material
and H. heilmannii successfully colonized.43
In a recent study,23 mouse inoculation was compared
to histologic examination of carbolfuchsin-stained slides. Of
70 pig stomachs examined, 54 were positive using the mouse inoculation
technique versus only 17 of the 70 stomachs examined by histology,
and only 14 of 70 positive using the rapid urease test.

Helicobacter are not easily demonstrated using hematoxylin-and-eosin
stain on histologic samples. To make Helicobacter organisms
more prominent, the Warthin-Starry silver stain is favored by
many researchers,34 but this procedure is more difficult
and more expensive than many other staining techniques, such as
carbolfuchsin staining.44 Other possible procedures
include Giemsa stain, acridine orange (which requires a fluorescent
microscope), and phase-contrast microscopic techniques.

There is a need for serological tests that would enable rapid
screening of pig herds for H. heilmannii. In addition,
culture techniques and PCR tests need to be developed.

Clinical disease

The strong association between H. pylori and gastritis,
gastroduodenal ulcers, and gastric cancer is well established
in human medicine.6 A matched control study has compared
H. pylori and H. heilmannii gastritis in humans.45
These researchers found that H. heilmannii colonization
was mainly focal and restricted to the antrum, compared to a more
diffuse pattern for H. pylori. Gastritis was significantly
milder in H. heilmannii compared to H. pylori-infected
patients.

Gnotobiotic pigs experimentally infected with H. pylori
do develop gastritis, which can lead to erosions and ulceration
in the glandular region of the stomach, generally in the junction
of the fundus and antrum.46 The inflammatory response
to H. heilmannii is similar to H. pylori, except
that the inflammation is distributed mainly in the fundus compared
to the cardia and antrum of H. pylori-infected pigs. The
vast majority of stomach ulcers in commercially reared swine occur
in the nonglandular pars oesophageal region, an area not suitable
for Helicobacter colonization. The pars oesophagea is lined
by stratified squamous epithelial cells and is normally inhabited
by various bacteria, including Lactobacillus and Bacillus
organisms.

In a recent study,33 gnotobiotic pigs were experimentally
infected with either fermentative bacteria such as Lactobacillus
and Bacillus, or H. heilmannii, and both groups
were fed a carbohydrate-enriched diet. Piglets infected with H.
heilmannii did not develop lesions in the pars oesophagea,
but the group infected with the fermentative bacteria did develop
epithelial damage in the pars oesophagea. The authors suggested
that the organic acids produced by bacteria such as Lactobacillus
and Bacillus and potentiated by high dietary carbohydrate
levels are important interactive factors for ulcer development
in the pars oesophagea.

On the other hand, the presence of H. heilmannii in
the glandular stomach has been found to be associated with ulceration
of the pars oesophagea in several epidemiological surveys.21,23,47,48
Barbosa, et al.,47 examined 32 pigs with grossly normal
mucosa and 32 pigs with chronic ulceration of the pars oesophagea.
Forty pigs (62.5%) were positive for H. heilmannii, and
of these positive animals, 67.5% had ulcers and only 32.5% were
normal. Of the 24 pigs negative for Helicobacter, five
(20.8%) had ulceration and 19 (79.2%) were normal. Queiroz, et
al.,23 examined 20 pig stomachs with ulcers, 30 stomachs
with parakeratosis lesions, and 20 normal stomachs. Helicobacter
were present in 100% of stomachs with ulcers, and 90% of stomachs
with parakeratosis, but only 35% of macroscopically normal stomachs.
These studies suggest a strong relationship between Helicobacter
infection and gastric ulceration.

Zoonotic potential

Molecular biological techniques have led to the conclusion
that the H. heilmannii organism isolated from a small proportion
of human gastritis patients is the same Helicobacter as
the organism widely distributed in the world swine population.
The potential for pig-to-human spread has been suggested.45,49
A recent survey of 177 patients with H. heilmannii infection
found that H. heilmannii infection was strongly associated
with contact with dogs, cats, cattle, or pigs.10 Contact
with pigs was found to be a greater risk factor than contact with
other animal species. People with pig contact were almost five
times more likely to be infected with H. heilmannii than
those without pig contact.

More studies must be performed to fully investigate the relationship
between H. heilmannii infection in pigs and humans, and
to determine how the organism might be transmitted. In addition,
the infection in humans and in pigs needs to be investigated to
determine the pathogenic significance of H. heilmannii
in both species.

The common gastric bacterium of humans (H. pylori)
does not naturally infect pigs but can be used to colonize the
pig's stomach experimentally.

The presence of Helicobacter can be detected by using
in situ urease mapping and histologic examination with Warthin-Starry
Silver stain.

Experimentally infected pigs have not developed gastric ulcers;
however, epidemiological studies have found a strong correlation
between the presence of H. heilmannii and ulceration of
the pars oesophagea.

H. heilmannii colonizes the fundic and/or pyloric
regions of the stomach, far removed from the pars oesophagea,
the ulcer site in pigs, and therefore it is difficult to explain
how the lesion could be caused by bacterial infection.

Acknowledgements

This work has been sponsored by Ontario Pork and the Ontario
Ministry of Agriculture Food and Rural Affairs.